Specific intrinsic membrane mechanisms controlling cytoplasmic Ca++ levels in vascular smooth muscle have remaized elusive due to contractile heterogeneity and difficulties in assigning specific cellular sites to the many identifiable intracellular CA++ compartments. While it is presumed that both sarcolemma and sarcoplasmic reticulum are involved in this regulation, the two membranes have not been convincingly separated to allow selective characterization of Ca++ kinetics of each. Quantitative assessment of Ca++ sequestering capacities of either membrane area alone as sole effector of relaxation is not successful, suggesting that additional mechanisms must be active to effectively lower the availability of activator Ca++. We propose that an additional mechanism may involve the Na+ pump, and that a predominance of sarcolemmal or sarcoplasmic reticulum control may actually determine the Ca++ requirements of a given tissue. This proposal will seek to: 1. separate sarcolemma from sarcoplasmic reticulum to achieve a study of each organelle's Ca++ sequestering and release capacity, 2. isolate a Ca++ + Mg++ ATPase using antibody-affinity column chromatography to determine the nature of the Ca++ ATPase activity responsible for active Ca++ sequestration, 3. fully characterize Rb86 uptake of renal, femoral, coronary and mesenteric arteries as a functional measure of the Na+ pump, 4. relate Rb86 uptake characteristics to the number of pump sites in these tissues, using 3H-ouabain binding both to intact tissue and subcellular fractions, 5. relate Rb86 data and 3H-ouabain data to K+-induced relaxation of the various arteries, 6. isolate and characterize Na+, K+-ATPase from these tissues using conventional techniques of differential ultracentrifugation as well as antibody affinity column chromatography. The correlation of these functional aspects of smooth muscle such as contractility and Rb86 uptake with Ca++ sequestering capabilities of sarcoplasmic reticulum and sarcolemma, Ca ATPase activities, and the characteristics of Na+, K+-ATPase will lead to an understanding of the intrinsic regulatory control mechanism of vascular smooth muscle, and enhance the concept that variations of these control mechanism of these tissues may play a role in determining Ca++ requirements for contractility.